U.S. patent application number 10/598693 was filed with the patent office on 2007-07-12 for ellipsometric biosensor comprising an amplification layer.
This patent application is currently assigned to OC OERLIKON BALZERS AG. Invention is credited to Johannes Edlinger, Andreas Eing, Dirk Hoenig, Max Wiki.
Application Number | 20070159629 10/598693 |
Document ID | / |
Family ID | 34919589 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070159629 |
Kind Code |
A1 |
Eing; Andreas ; et
al. |
July 12, 2007 |
Ellipsometric biosensor comprising an amplification layer
Abstract
A method for analyzing organic adsorbent layers (7) on a
substrate (4) comprises the steps of providing a substrate (4)
whose surface has an index of refraction equal or close to the
index of refraction of the organic adsorbent to be analyzed. On the
surface of the substrate (4) there is applied a layer system (5,6)
with at least one layer (5) with index of refraction of the
biological material and an organic adsorbent layer (7) on top of
the layer system. When acting polarized light upon the substrate a
change of polarization characteristics in reflection and/or
transmission is detected.
Inventors: |
Eing; Andreas; (Gottingen,
DE) ; Wiki; Max; (Ecublens, CH) ; Edlinger;
Johannes; (Feldkirch, AT) ; Hoenig; Dirk;
(Gottingen, DE) |
Correspondence
Address: |
PEARNE & GORDON LLP
1801 EAST 9TH STREET
SUITE 1200
CLEVELAND
OH
44114-3108
US
|
Assignee: |
OC OERLIKON BALZERS AG
P.O. Box 1000 Iramali 18
Balzers
LI
|
Family ID: |
34919589 |
Appl. No.: |
10/598693 |
Filed: |
March 7, 2005 |
PCT Filed: |
March 7, 2005 |
PCT NO: |
PCT/CH05/00136 |
371 Date: |
November 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60551290 |
Mar 8, 2004 |
|
|
|
Current U.S.
Class: |
356/369 |
Current CPC
Class: |
G01N 21/75 20130101;
G01N 21/211 20130101; G01N 2021/7773 20130101 |
Class at
Publication: |
356/369 |
International
Class: |
G01J 4/00 20060101
G01J004/00 |
Claims
1. A method for analyzing organic adsorbent layers on a substrate,
the method comprising the following steps: providing a substrate
with a surface, the substrate having an index of refraction equal
or close to the index of refraction of the organic adsorbent to be
analyzed applying a layer system to the surface of the substrate,
the layer system comprising at least one layer with index of
refraction significantly different from the index of refraction of
the biological material; applying the organic adsorbent layer on
top of the layer system act polarized light upon the substrate
detect in reflection and/or transmission the change of polarization
characteristics of the light acted upon the substrate.
2. Method according to claim 1 comprising the step of detecting the
reflection and/or transmission amplitude of the light acted upon
the substrate.
3. Method according to one of the previous claims comprising the
step of selecting the substrate material out of the group of glass
and plastic materials.
4. A method according to one of the previous claims characterized
in that said at least one layer is chosen out of the group of
Ta.sub.2O.sub.5, Nb.sub.2O.sub.5, TiO.sub.2, HfO.sub.2, ZrO.sub.2
MgF.sub.2.
5. Method according to one of the previous claims comprising the
step of optimizing the layer thickness distribution of the layer
system in order to provide optical response allowing for desired,
preferably maximum sensitivity with respect to changes in the
adsorbent layer to be analyzed.
6. Method according to one of the previous claims comprising the
step of selecting SiO.sub.2 as top layer forming the interface to a
contact layer and/or the adsorbent layer to be analyzed.
7. Method according to one of the previous claims comprising the
steps of applying a structured layer system thereby creating
multiple subareas with maximum sensitivity for different adsorbent
layer characteristics.
8. Method of performing imaging ellipsometry comprising the steps
of one of the methods according to one of the previous claims.
Description
TECHNICAL FIELD
[0001] This invention relates to biosensors for the detection of
adsorption and binding processes of biologically relevant molecules
at a surface.
BACKGROUND ART
[0002] The detection of binding reactions of biologically relevant
molecules, e.g. antibody-antigen reactions play an important role
in many biotechnology applications like pharmaceutical drug
screening. To monitor such reactions, photometric detection using a
fluorescent label attached to at least one of the involved
molecular species is common art.
[0003] However, besides the additional effort to label the
molecules, the presence of such markers is suspected to alter the
reaction kinetics. Therefore, marker-free detection schemes that do
not require the attachment of markers like fluorescent labels,
nanoparticles or radiating markers to the molecules of interest are
gaining increasing importance.
[0004] Several marker-free detection schemes have been developed
which allow a direct detection of a binding reaction or, more
generally, the adsorption or sedimentation of molecules at a
surface. The surface may be tailored to be chemically specific. In
the case of an immuno-reaction this may be accomplished by the
immobilisation of antibodies to a chemically pre-treated surface
and the subsequent binding of antigenes from a solution which is
probed with a suitable detection system. Detection may take place
with the sensing surface in contact to a solution or against air,
after prior removal of the solutions. Instruments that allow a
detailed monitoring of the adsorption kinetics are known, as well
as binary detection schemes where the absence or presence of a
certain molecular species is being probed.
[0005] In particular, optical detection techniques have been
applied successfully, allowing a remote, non-contact sensing. A
common feature of many of these techniques is a measurement of the
change of the intensities, the phase or state of polarization of
the probing light upon reflection at the examined surface or in
transmission or combinations thereof. This primary physical effect
is caused by the change of the optical thickness of the adsorption
layer, either due to a change in the dielectric properties of the
layer or the geometric layer thickness or both.
[0006] Ellipsometry is a well-known technique to determine the
thickness of an optical layer, or changes thereof during a growth
process. It sensitively measures the change of the state of
polarization when electromagnetic radiation is reflected or
transmitted by a sample. A classical embodiment of such an
apparatus is given by a light source that emits a collimated light
beam passing a variable polarisation controller given by the
combination of a linear polariser (P) and an compensator in the
form of a quarter-wave plate (C). The polarised light beam is
incident on the sample (S) under a known oblique angle, reflected
from the sample surface and analyzed by a second linear polarizer
(A) with the help of a photodetector. In this PCSA ellipsometer
setup the measurement may be done by changing the azimuths of the
components P and A, while the optical axis of C is kept at a
constant azimuth, e.g. at 45.degree. with respect to the plane of
incidence, until the photodetector receives a minimum of intensity.
The azimuthal angles of the components P, C and A for this
"nulling" condition may be used to calculate the ellipsometric
angles Delta and Psi which are specific for the optical parameters
of the sample at a given angle of incidence and wavelength of
light. Using a suitable optical model and numerical regression, the
quantities Delta and Psi may be recalculated in terms of the
thickness of the optical layer, or changes thereof during a growth
process.
[0007] Besides this classical Nulling Ellipsometer, many other
forms of ellipsometers have been realized, some of which measuring
only one of the two ellipsometric angles or systems that account
for depolarization effects.
[0008] The application of ellipsometry for monitoring of binding
reactions of biological molecules dates back to 1942 (A. Rothen, K.
Landsteiner, J. Exp. Med 76, 437 (1942)). The amount of adsorbed
biological material at a surface during a binding reaction may be
recalculated from the quantities Delta and Psi.
[0009] Prior art is also imaging ellipsometry (U.S. Pat. No.
5,076,696) which uses spatially resolving detector and imaging
optics to allow for a massively parallel measurement of
ellipsometric data, e.g. in the form of Delta and/or Psi maps. Such
maps may in turn be converted into surface maps of layer thickness,
optical index of refraction, chemical composition or the amount of
adsorped material. Frequently, biosensors are designed in the form
of an array of spots or wells to allow high throughput screening of
a multiple of molecular species simultaneously. Imaging
ellipsometry with its intrinsic parallel detection scheme may be
used avantageously as a detection technique for these so-called
biochips, microarrays or microplates (A. Eing, M. Vaupel, Imaging
Ellipsometry in Biotechnology, 2002, ISBN 3-9807279-6-3).
[0010] Imaging ellipsometry has been demonstrated with light
employed for the measurement impinging on the surface to be
measured coming from the ambient medium. Other measurement setups
are based on total internal reflection as described for example in
U.S. Pat. No. 6,594,011. Here the light from a light source is
directed through an internal reflection element to reflect off the
specimen to be detected.
[0011] As the amount of adsorbed material is usually very small,
equivalent to thickness changes in the range of nanometers or
below, and many interesting materials like proteins or DNA do not
exhibit a significant optical absorbance in the easily accessible
UV-VIS-NIR wavelength regime, very often the signal response for
optical detection is not high enough, thereby limiting
sensitivity.
[0012] Therefore, some imaging ellipsometers use surface plasmon
resonance (SPR) in order to increase the signal response for
optical detection. SPR uses a thin metal layer to allow the
excitation and propagation of surface plasmons. While one side of
the metal layer is in contact with a transparent support structure,
usually attached to a prism allowing to couple-in light under an
oblique angle, the other side of the layer is exposed to the
ambient medium. Changes in the optical index of refraction in the
ambient by the formation of an adsorbent layer are monitored as a
shift in the angle of incidence that generates surface plasmon
resonance, causing a change of reflected light intensity.
[0013] For SPR based sensors it is known that an intermediate
dielectric layer between the metal film and the probed surface may
act as a means to further increase the sensitivity, as for example
described in U.S. Pat. No. 5,999,148. This patent describes the
usage of such an intermediate layer including high-optical index of
refraction oxides and notes the importance of a certain layer
thickness to achieve the desired performance.
PROBLEM OF THE PRIOR ART TO BE SOLVED BY THE PRESENT INVENTION
[0014] Ellipsometry is used very successfully for thin film
applications, especially in the area of semiconductors. However, if
the adsorbent layer to be analyzed has an optical index of
refraction equal or close to the optical index of refraction of the
underlying substrate, the sensitivity of ellipsometric detection is
very limited. This is because in such cases the adsorbent layer
does not form an optical significant interface to the substrate, so
that no or only weak optical interference occurs at this interface.
A change in thickness of the adsorbent layer is therefore
comparable to a change in thickness of the underlying substrate and
merely results in a detectable phase shifting effect. Very often
glass or transparent plastic materials are the preferred substrate
materials to be used in order to measure biological materials.
Unfortunately, the optical index of refraction of biological
materials like proteins or DNA is close to that of glass or
transparent plastic materials, limiting the sensitivity of
ellipsometric detection for the case of organic material adsorbed
to such a substrate.
[0015] It is therefore the goal of the present invention to
disclose substrate samples providing improved optical response in
ellipsometric measurements, the improvements resulting from effects
other than surface plasmon resonances. Improvements in this context
means either increased sensitivity and/or better linearity and/or
increased dynamic range for the measurement of the adsorbent
layer.
DISCLOSURE OF THE INVENTION
[0016] The problem is solved by applying to the substrate an
amplification layer system comprising at least one dielectric layer
with an optical index of refraction significantly different from
the adsorbent layer to be detected. As a consequence at least one
optical significant interface is established between the adsorbent
layer and the substrate and optical interference effects get more
pronounced. The dielectric layer system can be chosen in such a way
that a change of thickness of the adsorbent layer results in
significant phase shifting and therefore leads to improved optical
response in ellipsometric measurement.
[0017] To optimize the design of the amplification layer system,
the response of the ellipsometric angles as a function of the
thickness of the adsorbent layer has to be calculated. The
calculation itself can be done by well-known procedures, as
described in R. M. A. Azzam, and N. M. Bashara, Ellipsometry and
Polarized Light, North Holland Press, Amsterdam 1977. In order to
establish increased sensitivity the goal of the optimisation is a
large slope of the measured ellipsometric angle (usually the Delta
is more sensitive for very thin films) as a function of layer
thickness of the adsorbent layer. A larger slope indicates a higher
sensitivity. However, at the same time the signal response at the
detector during the measurement procedure must also be sufficient.
Equations for the detector response of ellipsometers are also given
in R. M. A. Azzam, and N. M. Bashara, Ellipsometry and Polarized
Light, North Holland Press, Amsterdam 1977.
[0018] By balancing both signal response and Delta slope, an
optimum sensitivity for the practical measurement can be found for
a certain design of the intermediate layer system according to the
present invention.
[0019] If the object is to realize a linear response of the
biosensor in a certain thickness range of the adsorbent layer, the
layout of the amplification layer system can be modeled according
to the method described above with the optimization goal modified
in such a way, that designs providing linearity of optical response
are preferred.
[0020] In one embodiment of the present invention the amplification
layer system comprises just one single dielectric layer. The index
of refraction of this layer is either below or above the index of
the adsorbent layer. However in another embodiment of the present
invention the intermediate layer system comprises multiple layers
of materials with alternating index of refraction
[0021] Depending on the optical design of the intermediate layer
system the sensor works at various angles of incidence, including
but not necessarily TIR conditions.
[0022] The amplification layer system may comprise linker chemistry
and/or contact layers and/or activation layers and/or other
additional intermediate layers required to create desired surface
chemical properties.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Since the optimum layout of the amplification layer system
depends on the operation conditions of the biosensor, we describe
as an example a measurement set-up using a PCSA-ellipsometer for
the measurement of binding kinetic. Such an apparatus comprises a
light source that emits a collimated light beam passing a variable
polarization controller given by the combination of a first linear
polarizer (P) and an compensator in the form of a quarter-wave
plate (C). The polarised light beam is incident on the sample
substrate (S) under a known oblique angle, reflected from a sensing
surface of the sample substrate and analyzed by a second linear
polarizer (A) with the help of a photodetector.
[0024] For such measurements the sensing surface has to be in
contact with the solution of the molecular species to adsorb or
bind to it. Preferably this is in a flow cell that allows a
controlled flow of analyte over the surface, or in the well of a
microplate. The sensing surface forms the bottom of such a flow
cell or well in a microplate, with a beam of light propagating
through the substrate of the substrate sample to the sensing
surface and being reflected at the sensing surface.
[0025] In our example, during the measurement a light beam 1 enters
a coupling prism 2 and transmits through an optical contact layer 3
to the substrate 4 and the adsorbent layer 7 as is shown in FIG.
1.
[0026] The coupling prism 2 is used in order to illuminate the
sensing surface through the substrate 4 with an angle of incidence
which is above the critical angle of total internal reflection. As
the sensitivity of ellipsometry strongly depends on the angle of
incidence, it is favorable to get to higher internal angles by the
use of a coupling prism 2.
[0027] However, a direct illumination resulting in lower internal
angles without the need for a prism or other coupling device might
still lead to sufficient sensitivity for some applications by using
a proper amplification layer and would in this case be a preferred
solution. In our example the coupling prism 2 is made of BK7,
however any other transmitting glass or plastic types could be
used. In some cases it might be advantageous to use glass with low
stress birefringence in order to avoid depolarization effects.
[0028] The optical contact layer 3 is used to avoid additional
reflections form the interface between coupling prism 2 and
substrate 4. The optical contact layer 3 could be index matching
fluid such as for example index matching oil.
[0029] As can be seen from FIG. 1 the amplification layer system 5
according to the present invention is provided on the top surface
of the substrate 4. In our example the amplification layer system 5
comprises a single dielectric layer of a high-optical index of
refraction material which is deposited on a glass substrate. The
layer has a thickness that allows for a highly sensitive and linear
measurement of the organic adsorbent layer. The thickness of this
single dielectric layer is crucial to act as the amplification
layer and deduced from optical modeling as described above.
[0030] As an example we modeled a system which comprises a glass
substrate 4 with an optical index of refraction of 1.52, e.g. BK7,
an amplification layer system with a single dielectric layer with
an optical index of refraction of 2.2, e.g. Ta.sub.2O.sub.5 and
variable thickness of 0-150 nm. On top of this amplification layer
a 10 nm layer of SiO.sub.2 with an optical index of refraction of
1.46 is applied in order to provide a contact layer for the
adsorbent layer. (More details why such a contact layer is applied
are given below). The adsorbent layer has an optical index of
refraction of 1.5. The ambient medium is assumed to be water with
an optical index of refraction of 1.333, the wavelength is 632.8 nm
and the angle of incidence within the glass substrate is 60
degrees, requiring a prism or other optical coupler.
[0031] Table 1 gives a comparison of the ellipsometric performance
of a kinetics sensor with and without amplification layer as
simulated by optical modeling. The comparison is based on a Figure
of Merit (FOM) that takes into account the measuring and data
analysis process of a PCSA nulling ellipsometer as described above.
The definition is such that the sensor without amplification layer
has a FOM of 1. As may be seen from Table 1, a maximum FOM of
approx. 30 may be achieved for a 90 nm thick amplification layer,
corresponding to a 30-fold increase of sensitivity for the
detection of a thin layer of adsorbent molecules. Therefore the
optimum thickness for the amplification layer of this example is 90
nm, however a layer thickness from 70 nm to 100 nm shows good
performance as well. There are a number of details and alternatives
we discuss in the following:
[0032] a) Amplification Layer System
[0033] In our example as discussed above we used a thin layer of
Ta.sub.2O.sub.5 as amplification layer system. However other
materials such as Nb.sub.2O.sub.5, TiO.sub.2, HfO.sub.2 or
ZrO.sub.2 may equally be used. Nitrides like Si.sub.3N.sub.4 can
also be used and even Si at IR wavelengths. In principle, any other
typical thin film material having a optical index of refraction
above or below the index of the adsorbent layer can be used. One
example for a low index material would by MgF.sub.2.
[0034] In addition it is possible to optimize and use an optical
multilayer system as amplification layer system. Such a multilayer
system may comprise alternating high and low index layers. In order
to model such systems known transfer matrix methods can be applied.
In order to optimize such systems known optimization techniques
such as for example genetic algorithms or simulated thermal
annealing methods can be applied. Due to the increased number of
parameters (thicknesses of the layers) there is a high degree of
freedom for optimizing for desired optical responses. As an
example, we modeled the effect of increasing the layer thickness of
the SiO.sub.2 layer in the example above, which now not only acts
as a contact layer but effectively becomes a part of the
amplification layer structure. Starting with the optimized single
amplification layer of Ta2O.sub.5 with a thickness of 90 nm we now
vary the thickness of the former contact layer of SiO.sub.2 in the
range of 10 nm to 280 nm. The results for a Figure of Merit defined
as above are given in Table 2. It shows, that at a thickness of 250
nm for the SiO.sub.2 layer the double amplification layer structure
further increases the theoretical sensitivity for the binding of
the adsorbant layer by a factor of 3.
[0035] It should be noted that the overall reflection is a coherent
and incoherent superposition of reflected components at all
involved optical interfaces. Some of them have been omitted in FIG.
1 for the sake of clarity.
[0036] b) Contact Layer
[0037] In our first example as described we applied a 10 nm SiO2
layer on top of the amplification layer. This contact layer 6 was
applied to further facilitate the attachment of a chemically
specific sensing layer. Such a contact layer 6 may always be
applied on top of the amplification layer system 5, provided that
it is at least partly transparent and its optical effect is well
within the dynamic range of the sensor. In a preferred form, this
contact layer comprises a thin layer of SiO2 of a few nanometers,
followed by silane based linker chemistry and/or other intermediate
layers and finally the chemically sensitive layer, e.g. immobilized
antibodies.
[0038] On the other hand such a contact layer may be attributed as
well as a part of the amplification layer system. Then the
optimization according to the present invention takes the optical
effect of this contact layer into account. Sometimes the thickness
of the contact layer itself can be used as well as optimization
parameter, as was shown above.
[0039] c) Illumination Means
[0040] In our example we used a prism in order to provide
illumination above the critical angle of total internal reflection.
Instead of the prism alternatively a grating coupler on the bottom
side of the glass substrate 4 might be used.
[0041] d) Imaging Ellipsometry
[0042] Attaching this sensor to a flow cell integrated with an
ellipsometer allows a measurement of the thickness or surface
coverage of the adsorbant layer 7 forming from the solution of the
analyte, or a binding kinetics. By applying imaging ellipsometry
this biosensor is able to monitor multiple binding sites or spots
simultaneously in a massively parallel way.
[0043] e) Alternatives of Ellipsometer Setups and Applications
[0044] Alternative forms of this sensor include the incorporation
into microplates as the bottom plate, where each well forms an
individually enclosed biosensor, or an inverted design for usage
with microarrays in air, e.g. for high throughput screening, where
the light beam 1 is coming from the air side according to FIG. 2.
Again, the thickness of the amplification layer system 5 is
optimized by optical modeling. By compromising the maximum
performance it is also possible to design the sensor substrate for
usage in both liquid or air ambient.
[0045] f) Multiple Sub Areas
[0046] Another modification is to divide a single binding site into
multiple sub-areas each with maximum sensitivity for a certain
adsorbent layer thickness or surface coverage by compromising the
dynamic range or the linearity. This may be achieved by patterning
of the amplification layer and optimizing its thickness locally. In
a further modification, sub-areas that allow for an internal
calibration or acting as a reference channel to compensate the
effect of non-specific binding or temperature drift of the
ellipsometric signal may be included.
[0047] For the use with microplates or other applications requiring
a large area with multiple detection sites to be analyzed, a
possible modification would use local grating structures,
microprisms or embossed surface relief structure to couple-in the
light beam for each individual well or detection site.
[0048] g) Combination with Fluorescence Methods
[0049] Another modification is the combination with grating and
guided mode fluorescence based detection such as WO 95/33197 and
U.S. Pat. No. 2002/135780. The grating structure does not disturb
in case that the detection is done in off-resonance mode.
TABLE-US-00001 TABLE 1 thickness of amplification Figure of layer
(nm) Merit 0 1 5 3.1 10 7.2 20 16.0 30 22.2 50 27.8 70 29.4 90 29.7
110 29.4 130 27.8 150 21.4
[0050] TABLE-US-00002 TABLE 2 thickness of SiO.sub.2 layer Figure
of (nm) Merit 10 29.7 100 31.2 200 46.3 220 63.3 240 87.6 250 90.3
260 82.3 280 57.9
* * * * *